EP0786970B1

EP0786970B1 - Calcification-resistant bioprosthetic tissue and methods of making same

Info

Publication number: EP0786970B1
Application number: EP95938792A
Authority: EP
Inventors: Robert J. Levy; Eyal Lerner
Original assignee: University of Michigan
Current assignee: University of Michigan
Priority date: 1994-10-21
Filing date: 1995-10-20
Publication date: 2005-12-07
Anticipated expiration: 2015-10-20
Also published as: DE69534671D1; IL115708A; WO1996013227A1; CN1162252A; EP0786970A1; JPH10507673A; NZ296283A; CA2202247A1; ATE311832T1; AU713605B2; NO971744D0; US5674298A; CN1200655C; IL115708A0; NO971744L; ZA958900B; DE69534671T2; BR9509473A; AU4003795A; EP0786970A4

Abstract

Naturally-derived bioprosthetic materials are treated with epoxide crosslinking agents. In some embodiments, the tissue is crosslinked with low molecular weight epoxides in aqueous solutions at high pH levels. In other embodiments, the tissue is crosslinked at physiologic pH levels with epoxide crosslinking agents catalyzed with tertiary or quaternary amines, such as Tris or imidazole. In an advantageous embodiment, bioprosthetic tissue is crosslinked and derivatized with an anticalcification agent, such as a polyphosphonate anticalcification agent, using a polyphosphonate:polyepoxide monoadduct.

Description

This invention relates generally to materials which are resistant to in vivo calcification, and more particularly, to methods of preparing calcification-resistant biomaterials, suitable for implantation in a living being, using epoxide crosslinking agents.
More than 100,000 cardiac valve prostheses are placed in patients each year. Frequently, valve replacement surgery is the only means of treating cardiac valve disease. Currently used replacement valves include mechanical valves which may be composed entirely of a synthetic polymeric material such as polyurethane; bioprosthetic valves derived from bovine pericardium or porcine aortic valves; and aortic homografts.
Use of mechanical valves is frequently complicated by thrombosis and tissue overgrowth leading to valvular failure. Bioprosthetic heart valves have improved thrombogenicity and hemodynamic properties as compared to mechanical valve prostheses. However, calcification is the most frequent cause of the clinical failure of bioprosthetic heart valves fabricated from porcine aortic valves or bovine pericardium. Human aortic homograft implants have also been observed to undergo pathologic calcification involving both the valvular tissue as well as the adjacent aortic wall albeit at a slower rate than the bioprosthetic heart valves. Pathologic calcification leading to valvular failure, in such forms as stenosis and/or regurgitation, necessitates re-implantation. Therefore, the use of bioprosthetic heart valves and homografts has been limited because such tissue is subject to calcification. In fact, pediatric patients have been found to have an accelerated rate of calcification so that the use of bioprosthetic heart valves is contraindicated for this group.
Unfortunately, pathologic calcification also further complicates the use of synthetic vascular grafts and other artificial heart devices, such as ventricular assist systems, because it affects the flexibility of the synthetic polymers used to produce the devices.
The mechanism for pathological calcification of cardiovascular tissue is not fully understood. Generally, the term "pathologic calcification" refers to the undesirable deposition of calcium phosphate mineral salts. Calcification may be due to host factors, implant factors, and extraneous factors, such as mechanical stress. There is some evidence to suggest that deposits of calcium are related to devitalized cells, and in particular, cell membranes, where the calcium pump (Ca⁺²-Mg⁺²-ATPase) responsible for maintaining low intracellular calcium levels is no longer functioning or is malfunctioning. Calcification has been observed to begin with an accumulation of calcium and phosphorous, present as hydroxyapatite, which develops into nodules which can eventually lead to valvular failure.
The preparation of bioprosthetic tissue prior to implantation typically includes treatment to stabilize it against subsequent in vivo enzymatic degradation, typically by crosslinking molecules, particularly collagen, on and in the tissue. Various aldehydes have been used for this purpose, including glyoxal, formaldehyde, and glutaraldehyde. Glutaraldehyde, however, is the agent of choice. In addition to fixing the tissue, glutaraldehyde is a good sterilizing agent and it reduces the antigenicity of the tissue. To date, glutaraldehyde is the only effective crosslinking agent for preparing tissues for implantation that can be used at physiologic pH under aqueous conditions. Unfortunately, glutaraldehyde is now known to promote calcification. There is, thus, a need in the art for a means of crosslinking bioprosthetic tissue without promoting calcification.
Non-aldehyde crosslinking agents have been investigated, such as polyepoxides (e.g., polyglycerol polyglycidyl ethers sold under the trademark Denacol by Nagasi Chemicals, Osaka, Japan), but there have been no conclusive studies demonstrating efficacy of polyepoxide cross-linked tissues in vivo.
Research on the inhibition of calcification of bioprosthetic tissue has primarily focussed on tissue pretreatment with either detergents or diphosphonate anticalcification agents. Detergent pretreatment with noncovalently-linked detergents, such as sodium dodecyl sulfate (SDS), and a covalently bound detergent, such as amino oleic acid, have been demonstrated to be efficacious in materials exposed in circulating blood. However, both detergents and diphosphonates tend to wash out of the implanted bioprosthetic tissue with time due to blood-material interactions. Thus, these treatments merely delay the onset of the inevitable calcification process. Accordingly, there is also a need for a means of providing long-term calcification resistance for bioprosthetic heart valves and other implantable biomaterials or devices which are subject to in vivo pathologic calcification. In addition, detergents disadvantageously affect the tissue, resulting in a diminution of the collagen denaturation temperature, or shrink temperature (T,), which is an important measure of material strength, durability, and integrity. In some cases, use of detergents results in local toxicity. There is, thus, a need far an effective method of imparting anticalcification properties to bioprosthetic tissues which is not accompanied by the deleterious effects of detergents. All of the foregoing techniques still result in some degree of pathologic calcification in vivo as measured by calcium content of explanted specimens. There is, therefore, a need for a treatment that results in a greater level of calcification inhibition.
Systemic use of anticalcification agents, such as diphosphonates, results in significant side effects on bone, and overall, growth. There is, therefore, a need for site specific therapy for prevention of pathologic calcification which would offer low regional drug levels and minimal side effects.
US5080670 to Imamura and others discloses the use of polyepoxy compounds as hydrophilic cross-linking agents for bioprosethetic tissue to inhibit or prevent calcification thereof. More specifically, this document teaches the use of polyepoxy compounds such as "polyglycidyl ethers of polyglycerols, having a polymerization degree of 1 to 3 and/or polyglycidyl ethers of polyols" such as Denacol EX-313 or 314. The polyepoxy compounds disclosed by this document are therefore high molecular weight compounds.
US5296583 to Levy and others is fundamentally concerned with the inhibition or prevention of calcification of synthetic biomaterials being biocompatible elastomers, such as polyurethanes and polydimethylsiloxanes. The use of polyfunctional epoxides to form a bridge to irreversibly incorporate an anticalcification agent such as a polyphosphonate is discussed. An alternate description of this process is that a phosphonated epoxide (or monoadduct) is incorporated into the synthetic biomaterial during primary polymerization or into a pre-polymerised synthetic biomaterial.
It is, therefore, an object of this invention to provide biomaterials for implantation in a mammal which have increased resistance to in vivo pathologic calcification.
It is another object of this invention to provide biomaterials for implantation in a mammal which have a long-term, or prolonged, resistance to in viva pathologic calcification.
It is also an abject of this invention to provide biomaterials far implantation in a mammal which have localized calcification inhibition and, hence, avoid the toxic side effects associated with systemic administration of anticalcification agents.
It is additionally an object of this invention to provide methods of fabricating and/or treating biomaterials for implantation in a mammal using epoxy crosslinking agents to render the biomaterials more resistant to in vivo pathologic calcification.

SUMMARY OF THE INVENTION

The foregoing and other objects are achieved by this invention which provides, in one aspect thereof, a method of treating a biologically derived biomaterial for implantation in the interior of the body of a living being, the biomaterial being substantially insoluble in the interior of the body of a host living being, the method comprising the steps of incubating the biomaterial in a solution of an epoxide crosslinking agent at a suitable pH for a time sufficient to permit cross-linking of the biomaterial and blocking of calcification sites, characterised in that the epoxide crosslinking agent is condition: a polyfunctional epoxide that has at least two epoxy moieties per molecule, and the solution further contains a polyphosphonate having at least one functional group of a type which will react with an epoxy functionality such that a monoadduct is formed in said solution between these two compounds, the polyfunctional anticalcification agent being provided in a molar ratio of 1:2 so that there is an excess of free polyfunctional epoxide, the monoadduct-containing solution being diluted with water prior to incubating the biomaterial.
Preferably the biomaterial is pretreated with or stored in glutaraldehyde.
As used herein, the term "derivatized" means that an anticalcificatian agent, such as the aforementioned a polyphosphonate, is covalently attached to the surface of biomaterial tissue via an epoxy linkage.
The term "biomaterial" as used herein refers to collagenous material which may be derived from different animal, typically mammalian, species. The biomaterial is typically suitable for implantation, such as bioprosthetic tissue or the like, but the invention should not be limited thereby. Specific examples include, but arc not limited to, heart valves, particularly porcine heart valves; aortic roots, walls, and/or leaflets; bovine pericardium; connective tissue derived materials such as dura mater, homograft tissues, such as aortic homografts and saphenous bypass grafts; tendons, ligaments, skin patches, arteries, veins; and the like. Of course, any other biologically-derived materials which are known, or become known, as being suitable for in-dwelling uses in the body of a living being arc within the contemplation of the invention.
Any epoxy compound, which is preferably water-soluble and able to function as a calcium antagonist, is within the contemplation of the invention. Examples of suitable epoxide crosslinking agents, include without limitation, mono- or diepoxides, such as diglycidyl butanediol ester, ethanediol diglycidyl ester, erythritol anhydride (EDE), butanediol diglycidyl ether (GAB), and epichlorhydrin, as well as polyfunctional epoxides, such as the epoxides sold under the trademark Denacol by Nagasi Chemicals, Osaka, Japan. The Denacol epoxides are polyfunctional polyglycerol polyglycidyl ethers. For example, Denacol 512 has 4 epoxides per molecule and Denacol 521 has 5 epoxides per molecule. As used herein, the term "polyepoxide" means reactive polyfunctional epoxides having at least two epoxy moieties per molecule.
As used herein the term "polyphosphonate" includes compounds having at least two phosphonates per molecule. Such polyphosphonates are commercially available or can be synthesized by those of skill in the art. Exemplary polyphosphonates include 3-amino-1-hydroxypropane 1,1-diphosphonic acid (APD) and ethanehydroxydiphosphonate (EHDP). In certain embodiments, other polyphosphonates, such as aminomethyltriphosphonic acid and butylpentaphosphonic acid are preferred. Additional illustrative examples include, without limitation, hexamethylenediaminetetra(methylenephosphonic acid) and diethylenetriaminepenta(methylenephosphonic acid).
In an embodiment of the invention, a monoadduct of a polyphosphonate, and a polyepoxide is formed under conditions where an excess of polyepoxide remains. The excess epoxy crosslinks the tissue and the reactive epoxy functionality of the monoadduct attaches to amino groups of the tissue proteins so as to permanently bind covalently the phosphonate-containing adduct to the tissue.

BRIEF DESCRIPTION OF THE DRAWING

Comprehension of the invention is facilitated by reading the following detailed description, in conjunction with the annexed drawing, in which:
Fig. 1 is a graphical representation of the calcium content (µg/mg) of porcine aortic valve specimens, treated in accordance with methods of the invention, following 21 day subdermal implantation in rats;
Fig. 2 is a graphical representation of the calcium content (µg/mg) of porcine aortic valve specimens, crosslinked with high molecular weight polyepoxides in accordance with methods of the invention, following 21 day subdermal implantation in rats;
Fig. 3 is a graphical representation of the calcium content (µg/mg) of porcine aortic valve specimens exposed to a catalyst;
Fig. 4 is a graphical representation of the calcium content (µg/mg) of porcine aortic valve specimens exposed to epoxide crosslinking agents at high pH levels (10-11) in the presence of another catalyst;
Fig. 5 is a graphical representation of the collagen denaturation temperature (°C) for specimens of porcine aortic valves leaflets subjected to epoxide crosslinking agents at pH 10 or 11; and
Fig. 6 is a graphical representation of the collagen denaturation temperature (°C) for specimens of porcine aortic valves leaflets subjected to a polyepoxide crosslinking agent, Denacol, in the presence of a catalyst, at pH levels ranging from 6 to 12.

DETAILED DESCRIPTION OF THE INVENTION

Given below is a specific illustrative technique (see item II) for producing calcification-resistant bioprosthetic biomaterials in accordance with the principles of the invention. Although the examples given are primarily directed to the preparation of calcification-resistant heart valve components, the techniques described herein are applicable to the creation of any other device, prosthesis or implant comprising biomaterials of the type used for in-dwelling or surgically implanted devices.

A. High pH Processes

In one method, fresh bioprosthetic tissue is incubated in an aqueous solution of a water-soluble epoxide crosslinking agent at a pH of 10 or greater for a time sufficient to permit irreversible cross-linking and blocking of calcification sites. The concentration of the epoxide crosslinking agent, which may be a reactive polyfunctional epoxide, preferably ranges from about 0.01 M to 1.0 M, and more preferably from about 0.05 M to 0.5 M. The solution is buffered to a pH of greater than 10, and preferably in the range of 10 to 11, in any manner known in the art, illustratively with 0.5 M sodium borate.
In preferred embodiments, the incubation time is between about 24 hours and 21 days, typically 7 days. However, the length of time allotted for incubation in the embodiments described hereinabove is illustrative and can be varied by those of skill in the art. It should noted, however, that no deleterious effects on the bioprosthetic tissue have been observed during the suggested 7 day period. The incubation temperature may range from about 4° C to 63° C, so that proteins in the tissue will not become denatured. Preferably, the temperature is greater than about 20° C, and most preferably in the range of 25° C to 37° C.
Bioprostheses such as porcine aortic valve leaflets or bovine pericardium are typically stabilized and preserved in glutaraldehyde following harvesting, illustratively in a 0.2 % solution of glutaraldehyde in 0.05 HEPES buffer (N-2-hydroxyethylpiperzine-N'-2-ethanesulfonic acid available from Sigma Chemical Co., St. Louis, MO). Glutaraldehyde-preserved bioprostheses can then be stored at 4° C for prolonged periods of time. Glutaraldehyde-pretreated bioprosthetic tissue which is incubated in aqueous solutions of epoxide crosslinking agents exhibit superior calcification resistance.
In an example, porcine aortic valve leaflets are stabilized and preserved in a high pH (> 10, and preferably in the range of 10.5 and 11.5) solution of a low molecular weight epoxide, preferably 4 to 6 carbon atoms in core chain length, such as butanediol diglycidyl ether at 0.1 M, at a temperature of 25° C, for 7 days to confer stability and block calcification sites.

B. Physiological pH Processes

In another method, bioprosthetic tissue is exposed to an aqueous solution of a water-soluble epoxide crosslinking agent buffered to physiological pH, i.e., a pH in the range of 7.0 to 8.0, preferably 7.4. The crosslinking solution contains a tertiary or quaternary amine which acts as a "catalyst" to enable polyepoxide crosslinking of bioprosthetic tissue at physiologic pH under aqueous conditions. Suitable tertiary or quaternary amines useful in the practice of the invention, include without limitation, Tris(hydroxymethdyl) amino methanehydrochloride (Tris), and imidazole. In some embodiments, the catalyst also buffers the solution.
The epoxide crosslinking agent is present in the aqueous solution in a concentration range of 0.005 M to 0.5 M. The catalyst concentration is typically about 0.01 M, but may be varied relative to the concentration of epoxide crosslinking agent. Other reaction conditions, such as time and temperature, are in accordance with the high pH embodiments described hereinabove.
In a specific illustrative embodiment, a polyepoxide, such as Denacol 521 at 0.1 M concentration is buffered to pH 7.4 with an 0.01 M imidazole or Tris. A bioprosthetic tissue specimen is exposed to the epoxide solution for 7 days at 25° C.

II. Crosslinking and Derivatizing Bioprosthetic Tissue With Polyphosphonate:Polyepoxide Monoadducts

In accordance with an advantageous embodiment of the invention, bioprosthetic tissue can be simultaneously cross-linked and derivatized with a polyphosphonate:polyepoxide monoadduct. Polyphosphonate is combined neat with a stock of pure polyepoxide in a molar ratio of at least a 1:2, and preferably 1:10. Under these conditions, a monoadduct of the polyphosphonate:polyepoxide is preferentially formed, leaving excess unreacted polyepoxide. The monoadduct-containing mixture is diluted with water to a concentration in the range of 0.005 M to 0.5 M, and preferably about 0.1 M. In some embodiments, the monoadduct-containing mixture may be buffered to physiologic pH with the catalytic amines, Tris or imidazole, as described above.
Bioprosthetic tissue, either fresh or glutaraldehyde-pretreated, is exposed to the diluted monoadduct-containing mixture for a time sufficient to both crosslink and reactively bind polyphosphonates to the bioprosthetic tissue. More specifically, the monoadduct-containing mixture, which also contains excess unreacted polyepoxide, crosslinks the tissue with the unreacted polyepoxide while the reactive epoxy functionality of the monoadduct attaches to amino groups of the tissue proteins so as to permanently bind covalently the phosphonate-containing adduct to the tissue. The result is permanent calcification resistance.
In an illustrative embodiment of this aspect of the invention, 1 M APD is added to 10 M of a reactive polyepoxide, such as GAB, and allowed to stand for about 30 minutes to form a monoadduct. The monoadduct solution is diluted with water to 0.1 M concentration and buffered to a pH between 7.5 and 10. Fresh bioprosthetic tissue is exposed to the diluted monoadduct solution for a period of 24 hours to 21 days at a temperature of 25°C to confer crosslinking and derivatization.
It should be noted that the concentration range for the diphosphonate salt (in the pure acid form) is given for purposes of illustration only, and can be varied by those of skill in the art to optimize both binding and cross-linking. Further, the temperature and length of time allotted for incubation in the embodiments described hereinabove is illustrative and can be varied by those of skill in the art.
Moreover, while an aqueous solution is recommended for bioprosthetic tissue inasmuch as organic solvents have deleterious effects on biologically-based tissue, organic solvents are certainly within the contemplation of the invention. Isopropanol and ethanol, for example, have been used safely in connection with bioprosthetic tissues.

Experimental Section:

Bioprosthetic Tissue in Rat Subdermal Model

Bioprosthetic tissue samples in the form of bovine parietal pericardium from mature cows were obtained at slaughter and immediately placed in solutions of a low molecular weight epoxide, diglycidylbutanediol ester (GAB), and a high molecular weight epoxide, Denacol 521 (DEN), at pH levels of 10 and 11, for 7 days at 37° C. For comparison, some specimens were also incubated in 0.6% glutaraldehyde, at pH 11, for 7 days at 37°C.

The bovine pericardium samples were implanted in two subcutaneous pouches dissected in the ventral abdominal wall of weanling rats (male, CD, Sprague-Dawley, weighing 50-60 gm). After a period of time (21 days and 60 days), the tissue samples were removed and examined for calcification by measuring the level of Ca⁺² ions in the tissue. The results are reported below in Table I.

Epox.	pH	Phos.	Bound Agent at Implant	Implant (days)	ca ²⁺ ( µ g/mg)
GAB	11	--	---	21	19.0 ± 5.9
GAB	11	--	---	60	23.4 ± 10.6
GAB	10	---	---	21	61.3 ± 12.4
DEN	11	---	---	60	107.8 ± 12.7
GLT	11	---	--	21	63.5 ± 7.3
GLT	11	---	---	60	109.4 ± 14.6
Notes: GAB = Diglycidylbutanediol ester DEN = Denacol 521 (polyglycidyl ether-pentaexpoxide) GLT = Glutaraldehyde

Table I shows significant anticalcification inhibition for specimens of bovine pericardium samples treated in low molecular weight epoxides at high pH.
Bioprosthetic tissue samples in the form of porcine aortic valve leaflets were exposed to solutions of GAB and Denacol 521 at pH levels of 10 and 11, for 7 days at 25°C. Other samples were exposed to monoadduct-containing solutions of APD:GAB and APD:Denacol (1 M polyphosphonate to 10 M polyepoxide) at pH 10 for 7 days at 25° C.
Following exposure to the epoxide crosslinking agents, the leaflet samples were rinsed free of the crosslinking solution and implanted in two subcutaneous pouches dissected in the ventral abdominal wall of weanling rats. After 21 days, the tissue samples were removed and examined for calcification by measuring the level of Ca⁺² ions in the tissue. The results are shown on Fig. 1 which is a graphic representation of the calcium content, in µg/mg tissue, for each specimen type. Uncross-linked tissue (fresh) is shown for comparative purposes.
Fig. 2 is a graphical representation of the calcium content (µg/mg) of porcine aortic valve specimens, crosslinked with the high molecular weight polyepoxide, Denacol 521 in accordance with two methods of the invention. In the first method, specimens were exposed to Denacol 521 in an aqueous solution buffered to pH 7.4 with imidazole. In the second method, glutaraldehyde-pretreated specimens were exposed to an aqueous dilution of the monoadduct APD:Denacol 521 (1:10) buffered to pH 7.4 with imidazole. Control specimens are glutaraldehyde cross-linked in accordance with typical practice in the art. For comparative purposes, glutaraldehyde-pretreated specimens were exposed to the diphosphonate, APD, at pH 7.4. As shown in Fig. 2, following 21 days of implantation in rat subdermal pouches, the specimens treated in accordance with the methods of the present invention contained significantly less calcium than the control.
In order to demonstrate that the epoxide crosslinking agent is responsible for the calcification resistance, glutaraldehyde-pretreated porcine aortic valve leaflets were placed in aqueous solutions of imidazole at pH 7.4 and Denacol 521 buffered to pH 7.4 with imidazole at 25° C for 7 days. Glutaraldehyde-pretreated porcine aortic valve tissue comprises the control. Fig. 3 is a graphical representation of the calcium content (µg/mg) of the specimens explanted at 21 days. The imidazole-catalyzed Denacol solution produced significant reductions in calcium content.
Fig. 4 is a graphical representation of the calcium content (µg/mg) of porcine aortic valve specimens exposed to a low molecular weight epoxide (GAB) and a high molecular weight epoxide (Denacol) at high pH levels (10-11, buffered with NaOH) at 25°C for 7 days in the presence of 0.1 M Tris. The inhibitory effect of Tris catalysis is clearly seen when the results of Fig. 4 are compared to Fig. 1 and Table I.
Fig. 5 show data demonstrating collagen denaturation temperature or shrink temperature, T_S equivalent to that derived by using glutaraldehyde for porcine aortic valves leaflets subjected to GAB or Denacol at pH 10 or 11. The collagen denaturation temperature of fresh, untreated leaflets and glutaraldehyde-treated control leaflets are shown for comparison.
Furthermore, equally satisfactory shrink temperatures can be attained through using epoxide crosslinking agents at physiologic pH through the use of 0.01 M Tris or imidazole. Fig. 6 shows the shrink temperature of porcine aortic valve leaflets exposed for 7 days at 25°C to an aqueous solution of 0.1 M Denacol with 0.01 M imidazole, buffered with NaOH to various pHs ranging from 6 to 12. Glutaraldehyde-treated tissue has a shrink temperature at approximately 87°C.

Claims

A method of treating a biologically derived biomaterial for implantation in a living being, the method comprising the steps of incubating the biomaterial in a solution of an epoxide crosslinking agent at a suitable pH for a time sufficient to permit cross-linking of the biomaterial and blocking of calcification sites,
characterised in that
the epoxide crosslinking agent is a polyfunctional epoxide that has at least two epoxy moieties per molecule, and the solution further contains a polyphosphonate having at least one functional group of a type which will react with an epoxy functionality such that a polyphosphonate:polyepoxide monoadduct is formed in said solution between these two compounds, the polyfunctional anticalcification agent being provided in a molar ratio of at least 1:2 so that there is an excess of free polyfunctional epoxide, the monoadduct-containing solution being diluted with water prior to incubating the biomaterial.
The method of claim 1, characterised in that the polyfunctional anticalcification agent is provided in a molar ratio of 1:10.
The method of claim 1 or claim 2, characterised in that the polyfunctional epoxide is selected from the group consisting of diglycidyl butanediol ester, ethanediol diglycidyl ester, butanediol diglycidyl ether, polyepoxides and polyglycerol polyglicidyl ethers.
The method of any one of the preceding claims characterised in that the biologically derived biomaterial has been pretreated with or stored in glutaraldehyde.
The method of any one of the preceding claims characterised in that the pH is chosen to be in the range of 7.5 - 10.0.
The method of any one of the preceding claims characterised in that the monoadduct containing solution is diluted with water to a concentration in the range of 0.005 M to 0.5 M.
The method of claim 6 characterised in that the concentration of the monoadduct containing solution is 0.1 M.
The method of any one of the preceding claims characterised in that the diluted monoadduct containing solution is buffered with a catalytic amine to a physiological pH in the range of 7.0 -8-0.
The method of claim 8 characterised in that the catalytic amine is either Tris or imidazole.
The method of any one of the preceding claims characterised in that the time sufficient to permit cross-linking is between about 24 hours and 21 days.
The method of any one of the preceding claims characterised in that said biologically derived biomaterial is a bioprosthetic tissue selected from the group consisting of bovine pericardium, porcine heart valve leaflets, saphenous bypass grafts, aortic homografts and dura mater.
A biologically derived biomaterial in that said biologically derived biomaterial is a bioprosthetic tissue selected from the group consisting of bovine pericardium, heart valves including porcine heart valves; aortic roots, walls, and/or leaflets, homograft tissues including saphenous bypass grafts and aortic homografts, connective tissue derived materials including dura mater, and tendons, ligaments, skin patches, arteries and veins, and including a monoadduct of polyepoxide/polyphosphonate covalently bound to the biomaterial, said biomaterial being substantially insoluble in the interior of the body of a host living being and obtainable by a method in any preceding claims.